1. Field of the Invention
The present invention relates to counting of particles. The method is particularly useful in the counting of a sample of particles having a high density and variability in size.
2. Background Art
In many industrial and medical applications there is a need to determine the concentration of particles contained in a liquid sample solution. This sample solution generally contains one type of particle, which is of some minimum size and larger. These particles are counted by passing the sample solution fluid with the particles to be counted through an aperture, which has an electric current passing through it. When these particles go through the aperture an electrical pulse is generated whose peak voltage is proportional to the size of the particle. This analog particle voltage pulse is applied to a comparator circuit, which will generate a +5 volt level particle count pulse when the comparators threshold is exceeded. The particle count pulse signal is normally at 0 volts until the particle crosses the threshold and then goes to +5 volts for as long as the signal is above the threshold and returns to zero volts when the particle is no longer in the aperture sensing zone. This +5 volt particle count pulse then drives a counter, which is incremented for each particle count pulse on the leading or trailing edge.
This counting technique works very well for lower concentration samples but is very inaccurate when high concentration samples are counted. There is a tendency to under count these higher concentration samples. This under counting is mainly caused by particle coincidence in the aperture. Coincidence occurs when more than one particle is sensed in the aperture at the same time, only one particle is counted. This results in undercounting for high concentration samples.
Typically, to correct for this coincidence loss a coincidence correction algorithm is applied to the raw count to improve the count for high concentration samples. One of the most basic and effective statistically derived coincidence correction formulas is (Wynn and Hounslowxe2x80x94Coincidence correction for electrical-zone (COULTER COUNTER(copyright)) particle size analyzers, Powder Technology, 93, 163-175 (1997), see, in particular, p. 164, Formula 1. The entire contents of this reference are hereby incorporated by reference).                     TC        =                              1            Z                    ⁢                      ln            ⁡                          (                              1                                  1                  -                                      Z                    *                    RC                                                              )                                                          (        1        )            
TC=True coincidence corrected Count in counts/s
RC=Raw Count
ESV=Effective Sensing Volume in xcexcL
FR=Flow Rate in xcexcL/s
Z=ESV/FR in seconds
The value of Z is adjusted until the result is linear. Parameter Z is a function of the aperture size, flow rate, and average particle size.
FC=TC*(DR/FR)*CFxe2x80x83xe2x80x83(2) 
FC=Final Count in Counts/xcexcL
DR=Dilution Ratio
CF=Calibration Factor
This correction improves the count some but the loss at high concentrations is still too great and at higher concentrations the raw count will not increase with increasing concentration and may even recover lower counts. This coincidence correction formula is no longer effective when this happens. If the raw counts for these very high concentrations could be corrected then that would allow the coincidence correction algorithm to perform better. Another method is needed that will reduce the particle losses for higher concentration samples.
The sample may be further diluted and rerun to reduce the loss and improve the raw count but this will result in reduced lab throughput as the sample has to be run again. The instruments dilution ratio may be increased to reduce the loss and improve the raw count but this will result in longer count times, which will impact the instruments throughput. It also degrades the count accuracy for low count normal samples. This would not be a preferred method to improve the count linearity.
The optimum solution is to achieve an accurate and linear particle count for high and low concentration samples in the shortest amount of time. This is very difficult for some sample types such as white blood cells where the ratio of abnormal to normal cell concentrations is very high (450,000/5,000 or 90 to 1). The dilution is typically low (251 to 1) to improve the throughput and accuracy of white normals. This low dilution makes the high concentration whites very difficult to count.
Another method of count correction is well known in the industry (see U.S. Pat. No. 4,447,883 to Farrell et al, the entire contents of which are hereby incorporated by reference). This method calculates the average time between particles or total wait time of the counted particle stream and sets the corrected count equal to the inverse of the average wait time. The basic theoretical formula is that the frequency or counts per second is equal to the inverse of the average period of the particle pulse stream.
This method assumes that the effects of the aperture size, particle size and flight time can be ignored. This method operates based on the wait time containing information about lost counts due to coincidence. If two particles go through the aperture too close together, then one count is lost. However, the two particles take up to twice as long to go through the aperture. This reduces the Total Wait Time and causes the true count to be increased.
The Total Flight Time as measured in this method is corrupted with numerous coincidence events. Therefore the average flight time cannot be accurately determined from the Total Flight Time divided by the raw count.
The Total Wait Time is an accurate measurement of the time that no particle is present. Therefore the average wait time derived by dividing the Total Wait Time by the raw count is a substantially reliable measurement. Also the Total Weight Time can be calculated by measuring the total flight time and subtracting this from the total count time.
One key parameter is not handled very well in current methods including both methods mentioned above. This is the consideration of the effects that particle size have on proper coincidence correction. Both methods above would not work properly if different size particles were run through the aperture. The coincidence correction method uses a fixed average particle size to determine the effective sensing volume. If the particle size changes from sample to sample then the formula will no longer work properly. The wait time count correction method does not include the true Total Flight Time in the formula. Even though it measures the Total Flight Time it only uses this information to calculate the Total Wait Time. The Total Flight Time as measured is corrupted by coincidence and is not a good measurement of the true flight time. This method also will not perform well with different size particles. This is important for sample types such as white blood cells. White blood cells typically have a wide range of particle sizes. Clearly, the effects of particle size must be included in the correction methods to make them work properly at high concentrations with multiple size particles. Most count systems also generate a size histogram for the particles being counted. The average size of the population counted can be determined from the histogram.
Various methods for coincidence correction are described in Wales and Wilson, The Review of Scientific Instruments, Vol. 32, No. 10, p. 1132-1136 (1961), Princen and Kwolek, The Review of Scientific Instruments, Vol. 36, No. 5, p. 646-653 (1965), U.S. Pat. No. 4,580,093 to Feier et al, U.S. Pat. No. 5,247,461 to Berg et al, and U.S. Pat. No. 5,452,237 to Jones, Jr.
Most count correction methods rely on only one technique to perform the correction. It would be better to utilize more than one technique to allow for more gentle correction from each one with a more linear (accurate) final result.
This present invention relates to an improved particle count correction method with better particle count linearity (better accuracy) for samples with wide variation in particle sizes and wide variations in concentrations (high concentrations often produce lower accuracy).
This is accomplished by providing a method for counting particles, comprising the steps of successively passing multiple particles through a particle sensing zone; introducing a first signal into said particle sensing zone for a period of time; measuring a second signal emanating from said particle sensing zone, said second signal being caused by modulation of said first signal by said particles passing through said particle sensing zone; generating raw data using said second signal, said raw data correlating to a raw count of particles passing through said chamber, a wait time count and a size of each particle; and processing said raw data by using a true average flight time and a true average wait time to obtain a corrected count of particles.
In another embodiment, the invention is directed to an apparatus for counting particles in a sample, comprising one or more particle sensors, each sensor having a sensing zone; a particle delivery unit for delivering particles to at least one of said particle sensing zones, said particles passing through at least one sensing zone; a particle measuring unit for determining the size of particles passing through at least one of said particle sensing zones, said sensor generating a particle size signal, and for determining the number of particles that pass through at least one of said particle sensing zones in a given period of time, said particle sensor generating a particle number signal; a device for calculating the average flight time of said particles in said sample based on said particle size signal and said particle number signal; and a correcting unit for correcting an apparent particle count to an adjusted particle count by adding a true average flight time to a true average wait time to obtain a corrected count of particles.
In still another embodiment, the invention is directed to an apparatus for counting particles, comprising a chamber having an inlet, an outlet and a particle sensing zone located between said inlet and said outlet; a pump for passing a fluid containing particles into said inlet, through said particle sensing zone and out of said outlet; an electric source arranged to pass an electric current through said particle sensing zone; an electric current detector for measuring electric current as particles pass through said particle sensing zone, said detector generating raw data indicative of the number of particles passing through said particle sensing zone and indicative of the size of particles passing through said particle sensing zone; and a program for processing raw data from said detector, said program having the capability add true average flight time to average wait time to give a true average period value.
In yet another embodiment, the present invention improves the wait time count correction method by adding the true average flight time to the average wait time to give a true average period value which will yield a more accurate count for samples with a wide range of cell sizes such as white blood cells. This is more accurately an average period count correction method. The true average flight time is derived from the mean value of the size histogram of the sample population and a formula which relates the particle flight time to mean size. This formula is empirically derived from the counting instrument and is a function of aperture size, particle size, and flow rate.
This method also uses both the Average Period Count correction method and an enhanced coincidence correction formula to correct the raw count. Adjustment factors FTCF, BaseZ, and CCRFT are used in their respective methods to adjust the amount of correction each one has on the count.
Average Period Correction Method Formula:
APC=Average Period Count
CT=Count Time in seconds
RC=Raw Count for one CT or the average from multiple CT""s.
WTC=Wait Time Count for one CT or the average from multiple CT""s.
CTClk=Count Time Clock in counts per second
AFT=Average Flight Time in microseconds,
(Count conversion 10 is in units of counts per microsecond)
FTCF=Flight Time Correction Factor                     APC        =                  RC                                    WTC              +                              FTCF                *                                  (                                      RC                    *                    10                    *                    AFT                                    )                                                                    CTClk              *              CT                                                          (        3        )            
Enhanced Coincidence Correction Formula:
TC=True Count
APC=Average Period Count
BaseZ=Main factor for control of coincidence correction
CCRFT=Coincidence Correction Flight Time factor.
Z=BaseZ+(AFT*CCRFT) (includes correction for particle size)                     TC        =                              1            Z                    ⁢                      ln            ⁡                          (                              1                                  1                  -                                      (                                          Z                      *                      APC                                        )                                                              )                                                          (        4        )            